The invention is directed to the substrate specificity, synthetic scope, and efficiency of aldolase catalytic antibodies 38C2 and 33F12. More particularly, these antibodies are shown to catalyze intermolecular ketone-ketone, ketone-aldehyde, aldehyde-ketone, and aldehyde-aldehyde aldol addition reactions and in some cases to catalyze their subsequent dehydration to yield aldol condensation products. Substrates for intramolecular aldol reactions are also identified.
The aldol reaction is arguably one of the most important Cxe2x80x94C bond forming reactions employed in synthetic transformations. Traditionally, the aldol reaction has been a proving ground for the development of asymmetric synthetic strategies. In the 1980""s the aldol reaction experienced a renaissance with the development of numerous strategies to effect highly stereoselective aldols (For reviews of the adol reaction, see Heathcock, C. H. in Asymmetric Synthesis, Morrison, J. D., ed., Academic Press, New York, Vol. 3, 1984; Evans et al Topics in Stereochemistry 1982, 12, 1; Masamune et al. Angew. Chem. Int. Ed. Engl. 1985, 24, 1; Heathcock et al. Aldrichim. Acta 1990, 23, 99.; Heathcock, C. H. Science 1981, 214, 395.; Evans, D. A. ibid. 1988, 240, 420.; Masamune et al. Angew. Chem. Int. Ed. Engl. 1985, 24, 1.; Evans, D. A.; Nelson, J. V.; Taber, T. R. Top Stereochem. 1982, 13, 1.; Heathcock, C. H.; et al, in Comprehensive Organic Synthesis, Trost, B. M., Ed. (Pergamon, Oxford, 1991), Vol. 2, pp. 133-319; Peterson, I. Pure Appl. Chem. 1992, 64, 1821).
Generally, this has been most successfully achieved through the use of stoichiometric quantities of chiral auxiliaries. In recent years the design of stereoselective catalysts of the aldol reaction has become a topic of interest. Most notable of these approaches is the Carreira aldol reaction where a chiral Ti(IV) complex (2-10 mol %) catalyzes the enantioselective addition of 2-methoxypropene to aldehydes with 66-98% ee (Yanagisawa, A; Matsumoto, Y.; Nakashima, H.; Asakawa, K.; Yamamoto, H.J. Am. Chem. Soc. 1997, 119, 9319., and references therein; Bach, T. Angew. Chem. Int. Ed. Engl. 1994, 33, 417., and references therein; Yamada, Y. M. A.; Yoshikawa, N.; Sasai, H.; Shibasaki, M. Angew. Chem. Int. Ed. Engl. 1997, 36, 1871. (b) Carreira, E. M.; Lee, W.; Singer, R. A. J. Am. Chem. Soc. 1995, 117, 3649).
As a challenge to traditional organic methodology, the application of natural aldolase enzymes as synthetic catalysts has yielded numerous efficient syntheses of stereochemically complex molecules, particularly in the area of carbohydrate synthesis. Since no asymmetric catalysts exhibits the scope of reactivity required to meet every synthetic challenge there is a need for methodologies that allow for the development of asymmetric catalysts. This is true of both transition metal based as well as enzyme based catalysts. For example, while the Carreira Ti(IV) complex is limited in scope to the use of the enolate equivalent 2-methoxypropene, fructose 1,6-diphosphate aldolase is limited to the use of dihydroxyacetone phosphate as the aldol donor substrate (Gijsen, H. J. M.; Qiao, L.; Fitz,W.; Wong, C.-H. Chem. Rev. 1996, 96, 443. (b) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem. Int. Ed. Engl. 1995, 34, 412-432. (b)Henderson, I.; Sharpless, K. B.; Wong, C.-H. J. Am. Chem. Soc. 1994, 116, 558. (c) Wong, C.-H.; Whitesides, G. M. Enzymes in Synthetic Organic Chemistry (Pergamon, Oxford, 1994); Bednarski, M. D., in Comprehensive Organic Synthesis, Trost, B. M., Ed (Pergamaon, Oxford, 1991), vol. 2, 455; Gijsen, H. J. M., Wong, C.-H. ibid. 1995, 117, 2947; Wong, C.-H. et al. ibid. 1995, 117, 3333; Chen, L.; Dumas, D. P., Wong, C.-H. ibid. 1992, 114, 741).
To address the problem of the de novo generation of protein catalysts of the aldol reaction, we recently described the development of two aldolase catalytic antibodies 38C2 and 33F12. These antibodies were raised against the xcex2-diketone hapten 1 which served as a chemical trap to imprint the lysine-dependent class I aldolase mechanism in the active site of the antibody. The suggested mechanism for the selection process of antibodies 38C2 and 33F12 during immunization is shown in FIG. 6. The xcex5-amino group of the lysine residue reacts with a carbonyl function of the xcex2-diketone moiety of 1 to form a xcex2-keto hemiaminal followed by dehydration to give a xcex2-keto imine that finally tautomerizes into a stable enaminone 2. Consequently, the hapten is now covalently bound in the binding pocket. The mechanistic similarity between this stoichiometric reaction and the accepted enamine mechanism of class I aldolase enzymes has been discussed in detail elsewhere (Wagner, J.; Lerner, R. A.; Barbas III, C. F. Science 1995, 270, 1797. (b) Zhong, G.; Hoffmann, T.; Lerner, R. A.; Danishefsky, S.; Barbas III, C. F. J. Am. Chem. Soc. 1997, 119, 8131. (c) Barbas III, C. F.; Heine, A.; Zhong, G.; Hoffmann, T.; Gramatikova, S.; Bjxc3x6rnestedt, R.; List, B.; Anderson, J.; Stura, E. A.; Wilson, E. A.; Lerner, R. A. Science 1997, 278, 2085).
The formation of the enaminone has been monitored by UV spectroscopy (with hapten 1: 1max=318 nm, xcex5xcx9c15000) and is complete within seconds to a few minutes, depending on whether antibodies were incubated with hapten 1, or other diketones such as 2,4-pentanedione or 3-methyl 2,4-pentanedione. Antibodies 38C2 and 33F12 have been previously shown to catalyze aldol reactions of some aliphatic ketones donors with two different aldehyde acceptors having a 4-acetanilide substituent in the xcex2-position as well as intramolecular aldol reactions that allowed for our recent antibody catalyzed synthesis of the Wieland-Miescher ketone (Zhong et al., ibid). Moreover, both antibodies were found to catalyze the decarboxylation reactions of aromatic xcex2-keto acids by the formation of a Schiff base between the xcex5-amino group of the lysine residue and the keto group of the substrate (Bjxc3x6rnestedt, R.; Zhong, G.; Lerner, R. A.; Barbas III, C. F. J. Am. Chem. Soc. 1996, 118, 11720).
What is needed is antibodies which can catalyze many aldol addition reactions with varying substrates producing desired enantiomeric outcomes and in some cases to catalyze their subsequent dehydration to yield aldol condensation products.
The invention is directed to the use of catalytic antibodies for catalyzing aldol condensation reactions and retroaldol reactions.
One aspect of the invention is directed to a method for catalyzing an aldol condensation between an aldol donor substrate and an aldol acceptor substrate for producing a xcex2-hydroxy ketone. A catalytically effective amount of a catalytic antibody having aldol addition activity or of a catalytically active molecule containing an antibody combining site portion of the catalytic antibody is admixed with sufficient amounts of the aldol donor substrate and aldol acceptor substrate in a reaction medium for producing a reaction admixture. The aldol donor substrate is of a type which includes a reactive carbonyl group and an unbranched carbon adjacent to the carbonyl group. The catalytic antibody or the catalytically active molecule is of a type which includes a lysine residue which forms a Schiff""s base intermediate with the reactive carbonyl group of the aldol donor substrate. The above reaction admixture is then maintained for a period of time sufficient for the catalytic antibody or catalytically active molecule to catalyze the aldol condensation between the aldol donor substrate and the aldol acceptor substrate for producing the xcex2-hydroxy ketone. The aldol donor substrate is either a ketone donor substrate or an aldehyde donor substrate. The aldol acceptor substrate is either a ketone acceptor substrate or an aldehyde acceptor substrate. However, the following proviso applies: if the aldol donor substrate is the ketone donor substrate and the aldol acceptor substrate is the aldehyde acceptor substrate, then the ketone donor substrate is not an unfunctionalized open chain aliphatic ketone. In a preferred mode of this aspect of the invention, the aldol acceptor substrate is an aldehyde acceptor substrate and the aldol donor substrate is an ketone donor substrate selected from the group consisting of aliphatic cyclic ketones, functionalized open chain aliphatic ketones, and functionalized cyclic ketones. The aldol condensation may be intermolecular or intramolecular. If it is intramolecular, both the aldol donor substrate and the aldol acceptor substrate form a single reactant molecule and the aldol condensation causes a cylization of the single reactant molecule for forming a cyclic xcex2-hydroxy ketone. The substrates may be heterogeneous, i.e., the donor and acceptor differ from one another, or homogeneous, i.e., the donor and acceptor are identically the same and are a single ketone reactant molecule.
Optionally, an addition step may be added to the above method wherein the above reaction admixture is maintained for a further period of time in the presence of the catalytic antibody or the catalytically active molecule for converting the xcex2-hydroxy ketone to a xcex2-unsaturated ketone product by an elimination reaction.
Alternatively, a different additional step may be added to the above method wherein the xcex2-hydroxy ketone is converted to a dihydroxy product by reduction.
In an alternative mode of the invention, the aldol donor substrate is represented by the following structure: 
The aldol acceptor substrate is represented by the following structure: 
The xcex2-hydroxy ketone is represented by the following structure: 
In the above structures, R1 is a radical selected from the group consisting of (FG)-alkyl, (FG)-alkenyl, and (FG)-aryl; R2 is a radical selected from the group consisting of H, OH, and F; X is a radical selected from the group consisting of NCH3, O, S, CH2 and C6H4; and FG is a radical selected from the group consisting of OH and OCH3.
Another aspect of the invention is directed to another method for catalyzing an aldol condensation between an aldol donor substrate and an aldol acceptor substrate for producing a xcex2-hydroxy ketone. The aldol donor substrate is either a ketone donor substrate or an aldehyde donor substrate substrate. The aldol acceptor substrate is either a ketone acceptor substrate or an aldehyde acceptor substrate. A catalytically effective amount of a catalytic antibody having aldol addition activity or of a catalytically active molecule containing an antibody combining site portion of the catalytic antibody is then admixed with sufficient amounts of the aldol donor substrate and of the aldol acceptor substrate in a reaction medium for producing a reaction admixture. The catalytic antibody or the catalytically active molecule is of a type which includes a lysine residue which forms a Schiff base intermediate with the aldol donor substrate. The aldol donor substrate is unbranched at a non-bond-forming xcex1 position. The above reaction admixture is then maintained for a period of time sufficient for the catalytic antibody or the catalytically active molecule to catalyze the aldol condensation between the aldol donor substrate and the aldol acceptor substrate for producing the xcex2-hydroxy ketone and for converting the xcex2-hydroxy ketone to a xcex2-unsaturated ketone product by an elimination reaction.
Another aspect of the invention is directed to a method for catalyzing a retroaldol reaction for converting xcex2-hydroxy ketone into a first and a second carbonyl product. The first and second carbonyl products are independently either a ketone product or an aldehyde product. A catalytically effective amount of a catalytic antibody having aldol addition activity or of a catalytically active molecule containing an antibody combining site portion of the catalytic antibody is admixed with the xcex2-hydroxy ketone in a reaction medium for producing a reaction admixture. The catalytic antibody or the catalytically active molecule is of a type which includes a lysine residue which forms a Schiff base intermediate with the first carbonyl product. The first carbonyl product is unbranched at an xcex1 position. The above reaction admixture is then maintained for a period of time sufficient for the catalytic antibody or the catalytically active molecule to catalyze the retroaldol reaction for converting the xcex2-hydroxy ketone to the first and second carbonyl products. In one mode of this aspect of the invention, the first and second carbonyl products may each be ketone products. In another mode of this invention, the first and second carbonyl products are each aldehyde products. And, in a further mode of this invention, the first carbonyl product is the aldehyde product and the second carbonyl product is the ketone product. The xcex2-hydroxy ketone may be either open chained or cyclic. If the xcex2-hydroxy ketone is cyclic, then the retroaldol reaction opens the cyclic xcex2-hydroxy ketone for forming a single open chain product containing both the first and second carbonyl products as a single product molecule. In another mode of this aspect of the invention, the retroaldol reaction is a reverse self-aldol condensation wherein the first and second carbonyl products are identical to one another. In a preferred mode of this aspect of the invention, the xcex2-hydroxy ketone is represented by the following structure: 
The first carbonyl product is represented by the following structure: 
The second carbonyl product is represented by the following structure: 
In the above structures, R1 is a radical selected from the group consisting of (FG)-alkyl, (FG)-alkenyl, and (FG)-aryl; R2 is a radical selected from the group consisting of H, OH, and F; X is a radical selected from the group consisting of NCH3, O, S, CH2, and C6H4; and FG is a radical selected from the group consisting of OH and OCH3.